With more than 380 million people currently affected, and an estimated 590 million by 2035, diabetes is a pandemic. Over time, 30-40% of diabetic patients develop nephropathy (DN), the leading cause of end stage renal disease (ESRD) requiring dialysis or kidney transplantation to sustain the patient's life. Lack of mechanistic aspects of DN pathogenesis is a significant impediment to rational development of new therapeutic treatments for preventing or delaying the progression to diabetic ESRD. We have recently demonstrated that collagen IV, a major component of renal extracellular matrix (ECM), is functionally damaged by hypochlorous acid (HOCl) in diabetic kidney, thus defining a new paradigm for investigating the pathogenic mechanisms underlying ECM lesions in diabetes. We propose to determine how this oxidative damage occurs and contributes to DN susceptibility and pathogenesis. Our central hypothesis is that pro-oxidative environment in diabetes causes excessive production of HOCl by a molecular complex discovered in our laboratory, which includes ECM-bound enzyme peroxidasin (PXDN), hypohalous acids (HOCl/HOBr) and extracellular chloride. The high levels of HOCl cause modification of collagen IV and cellular phospholipids at cell-ECM interface in renal glomerulus, thus perturbing collagen IV network stability and interaction with glomerular cells. This HOCl-derived damage contributes to progressive DN lesions, whereas inhibition of the damage ameliorates DN progression. The proposal will utilize the DN-prone and DN-resistant diabetic mouse models, conventional and imaging mass spectrometry, and pyridoxamine, an investigational drug, now in phase 3 clinical trials in DN, which is a scavenger of HOCl in vitro and in vivo. The proposal seeks 1) to determine how PXDN-mediated level of HOCl is affected by diabetic milieu; 2) to determine mechanism whereby modification of renal collagen IV by HOCl disrupts integrin-dependent cell-ECM interactions and compromises network stability in DN; and 3) to identify HOCl-derived chlorinated lipids and determine their effects on glomerular cells and contribution to glomerular lesions in DN. It is anticipated that the findings will give insight into mechanisms of DN susceptibility and pathogenesis and will establish a platform for the development of new drug candidates to slow DN progression.